A considerable number of known, naturally occurring small and medium-sized cyclic peptides as well as some of their artificial derivatives and analogs (in the context of the present application the term "peptide" also comprises peptide derivatives and analogs containing at least one peptide linkage) possessing desirable pharmacological properties have been synthesized up to now. However, wider medical use is often hampered by the relative complexity of their synthesis and purification. Therefore, improved methods for making these compounds are desirable.
An example for these compounds is the vasopressin analog desmopressin, a valuable medicine for the management of such ailments as diabetes insipidus and nocturnal enuresis.
In terms of peptide synthetic methodology, two major synthetic techniques dominate current practice. These are synthesis in solution (homogeneous phase) and synthesis on solid phase. In both, the desired peptide compound is created by the step-wise addition of amino acid moieties to a growing peptide chain. Larger peptide fragments can be coupled at various stages in the synthesis when working in homogeneous solution. However, a problem exists in the art for the preparation of cyclic peptide compounds based on disulfide links because separate operations are required before purifying the end product, which increases expense and may effect final product quality and quantity.
Oxidative cyclization of protected or non-protected sulfydryl groups with formation of disulfide structures usually is carried out as the final synthetic step, the reason being, inter alia, the substantial thermal and chemical lability of the disulfide linkage. An example of such a process is found in U.S. Pat. No. 4,271,068, "Process for the Manufacture of Cystine Containing Peptides" (the contents of which are incorporated by reference). This oxidation of open-chain peptides containing free and/or certain types of protected sulfhydryl groups with iodine in, e.g., methanol or acetic acid is further explained in the CRC Handbook of Neurohypophyseal Hormone Analogs, Vol. 1, Part 1; Jost, K., et al. Eds., CRC Press, Boca Raton, Fla. 1987, p. 31 and Table 2.
Iodine, however, is not without drawbacks as a cyclization agent (see Sieber, P., et al., 1980, Helv. Chim. Acta, 63:2358-2363; Cavelier, F., et al., 1989, Bull. Soc. Chim. France, p. 788, and literature cited therein). For instance, tyrosine moieties present in peptide substrates are at risk of being iodinated, making the balance between full conversion of starting materials and minimizing side reactions a delicate one, which, in turn, impacts product purity.
The purity of a peptide has several aspects. One is purity on an active-compound concentration scale. This is represented by the relative content of the pharmacologically active compound in the final product which should be as high as possible. Another aspect is the degree of absence of pharmacologically active impurities which, though present in trace amounts only, may disturb or even render useless the beneficial action of the peptide when used as a therapeutic. In a pharmacological context both aspects have to be considered.
As a rule, purification becomes increasingly difficult with larger peptide molecules. In homogeneous phase synthesis (which is the current method of choice for industrial production of larger amounts of peptides) repeated purification required between individual steps provides a purer product but low yield. Thus, improvements in yield and purification techniques at the terminal stages of synthesis are needed.
A variety of methods, most often in combination, have been used to purify cyclic small and medium size peptides obtained by oxidative cyclization with ferricyanide, i.e. ion exchange chromatography on a weakly acidic cation exchange chromatography resin (L. Juliano, et al., 1990, Proceedings of the Eleventh American Peptide Symposium, J. E. Rivier and G. R. Marshall, Eds., p. 955; Escom, Leiden, NL).
Another complicating factor in known synthesis routes is the possibility of interaction between the desired cyclic disulfide and inorganic sulfur compounds used for reducing excess iodine at the end of the reaction, such as sodium dithionite or sodium thiosulfate. Such reducing sulfur-containing compounds may interact with the disulfide linkage which is sensitive to nucleophilic attack in general.